小剂量衣霉素诱导的内质网应激预处理对大鼠缺血再灌注心肌局部炎症的影响

Preconditioning with endoplasmic reticulum stress induced by low dose tunicamycin attenuates myo- cardial inflammation in myocardial ischemia- reperfusion injury

目的观察小剂量衣霉素诱导内质网应激预处理对缺血再灌注损伤（I／R）大鼠心肌局部炎症的影响。方法40只成年雄性SD大鼠，随机分为假手术组（Sham组）、I／R组、单纯衣霉素处理组（TM组）及衣霉素＋I／R组（TM＋VR组），每组各10只。I／R组及TM＋I／R组通过结扎左冠状动脉前降支并再灌注的方法建立I／R模型，TM组及TM＋I／R组模型建立前30min腹腔注射衣霉素0．6mg／kg，分别于开胸后和再灌注2h取颈动脉血，使用比色法检测血清肌酸激酶同工酶（CK-MB）及乳酸脱氢酶（LDH）活性。再灌注2h后处死大鼠摘取心脏，使用原位缺口末端标记法（TUNEL）法观察心肌细胞凋亡，Westernblot法检测心肌半胱氨酰天冬氨酸特异性蛋白酶（Caspase）-12、核因子（NF）-κBp65蛋白的表达。结果Sham组、TM组CK-MB、LDH活性再灌注2h时与开胸后比较变化差异无统计学意义（P〉0．05），I／R组及TM＋I／R组CK—MB、LDH活性均较开胸后上升，两组间比较，VR组高于TM＋I／R组（P〈0．05）；Sham组、TM组、L／R组和TM＋I／R组细胞凋亡指数依次为（2．4±0．3）％、（7．7±0．6）％、（29．4±3．1）％和（12．8±1．9）％。I／R组、TM＋I／R组与Sham组比较，细胞凋亡指数较高，差异有统计意义（P〈0．05），L／R组与TM＋I／R组比较更为显著（P〈0．05）；再灌注2h后Sham组心肌组织Caspase-12、NF-κBp65蛋白呈低表达，I／R组、TM组和TM＋I／R组中均呈高表达，与Sham组比较差异有统计学意义（P〈0．05），其中TM组和TM＋I／R组均低于I／R组，差异有统计学意义（P〈0．05）。结论小剂量衣霉素诱导内质网预处理可能通过减轻缺血再灌注心肌的内质网应激，降低NF-κB通路激活，减轻心肌局部炎症，从而产生一定的心肌保护效果。

Objective To evaluate the protective effects of preconditioning with endoplasmic retic- ulum stress （ERS） induced by tunicamycin （TM） against myocardial inflammation in myocardial ischemia reperfusion （I/R） injury. Methods Forty Sprague - Dawley rats were randomly divided into four groups ： Sham, TM, I/R, and TM ＋ I/R. The model of I/R were established by occlusion of the left anterior de- scending coronary artery for 0. 5 h followed by reperfusion for 2 h in I/R group and TM ＋ I/R group. TM group and TM ＋ I/R group were treatmented by TM 0. 6 mg/kg at 30 min before model established. Blood samples were withdrawn from carotid artery after thoracotomy and 2 h after reperfusion, to detect the levels of creatine kinase isozyme （ CK - MB） and lactic dehydrogenase （LDH）. The protein expression of cystei- nyl aspartate-specific protease （Caspase） - 12 and nuclear factor （NF） - κB p65 in heart at the end of the I/R were examined by Western blotting in all groups. The myocardial apoptosis index （AI） using terminal deoxynucleotidyl nick - end labeling were observed in all groups. Results The levels of CK - MB and LDH, AI, and expressions of Caspase - 12 and NF - κBp65 protein in I/R group [ （546 ±69） U/L, （2 094 ± 328） U/L, （29.4 ±3.1 ） %, 0. 461±0. 046, and 0. 287 ±0. 032 respectively ] and TM ＋ I/R group [（977±103） U/L, （3 198±417） U/L, （12.8±1.9）%, 0.269 ±0.041, and 0. 153 ±0.031respectively] were higher than Sham group [ （559 ± 81 ） U/L, （2 459 ± 231 ） U/L, （2.4 ±0. 3 ） %, 0. 067 ±0. 009, and 0. 049 ±0. 006 respectively] and TM group [ （572 ±81） U/L, （2 335±241） U/L, （ 7. 7 ± 0. 6 ） %, 0. 149 ± 0. 031, and 0. 082± 0. 010 respectively ] at 2 h after reperfnsion （ P 〈 0. 05 ）. All observed indexes in I/R group were higher than TM ＋ I/R group at 2 h after reperfusion（ P 〈 0. 05 ）. Conclusion Preconditioning with ERS induced by low dose TM attenuates myocardial inflammatory, which may be associated with the reduced I/R injury in rats.